Investigation of antibacterial and anticancer effects of novel niosomal formulated Persian Gulf Sea cucumber extracts

Pharmaceutical companies worldwide are scrambling to develop new ways to combat cancer and microbiological pathogens. The goal of this research was to investigate the antibacterial, anticancer, and apoptosis effects of novel niosomal formulated Persian Gulf Sea cucumber extracts (SCEs). Sea cucumber methanolic extracts were prepared and encapsulated in niosome nanoparticles using thin-film hydration. The compound was made up of Span 60 and Tween 60 blended with cholesterol in a 3:3:4 M ratios. Characterization of niosome-encapsulated SCE evaluated by scanning electron microscopy and transmission electron microscopy. The disk diffusion method and microtiter plates were used to investigate the antimicrobial activity. The effect of niosome-encapsulated SCE on cell proliferation and apoptosis induction was studied using MTT and Annexin V, respectively. The expression of apoptosis-related genes, including Bax, Fas, Bax, Bak, and Bcl2, was studied using quantitative real-time PCR. Niosome-encapsulated SCE with a size of 80.46 ± 1.31 and an encapsulation efficiency of 79.18 ± 0.23 was formulated. At a concentration of 100 μg/ml, the greatest antimicrobial effect of the niosome-encapsulated SCE was correlated to Staphylococcus aureus, with an inhibition zone of 13.16 mm. The findings of the study revealed that all strains were unable to produce biofilms at a concentration of 100 μg/ml niosome-encapsulated SCE (p < 0.001). The survival rate of cancer cells after 72 h of exposure to niosome-encapsulated SCE was 40 ± 3.0%. Encapsulated SCE in niosomes inhibited cell progression in MCF-7 cells by increasing G0/G1 and decreasing S phase relative to G2/M phase; as a result, it activated the apoptosis signaling pathway and led to the induction of apoptosis in 69.12 ± 1.2% of tumor cells by increasing the expression of proapoptotic genes (p < 0.001). The results indicate that sea cucumber species from the Persian Gulf are a promising source of natural chemicals with antibacterial and anticancer properties, paving the path for novel marine natural products to be discovered. This is the first demonstration that niosome-encapsulated SCE contains antibacterial and anticancer chemicals that, according to their specific characteristics, boost antitumor activity.

Pharmaceutical companies worldwide are scrambling to develop new ways to combat cancer and microbiological pathogens. The goal of this research was to investigate the antibacterial, anticancer, and apoptosis effects of novel niosomal formulated Persian Gulf Sea cucumber extracts (SCEs). Sea cucumber methanolic extracts were prepared and encapsulated in niosome nanoparticles using thin-film hydration. The compound was made up of Span 60 and Tween 60 blended with cholesterol in a 3:3:4 M ratios. Characterization of niosome-encapsulated SCE evaluated by scanning electron microscopy and transmission electron microscopy. The disk diffusion method and microtiter plates were used to investigate the antimicrobial activity. The effect of niosome-encapsulated SCE on cell proliferation and apoptosis induction was studied using MTT and Annexin V, respectively. The expression of apoptosis-related genes, including Bax, Fas, Bax, Bak, and Bcl2, was studied using quantitative real-time PCR. Niosome-encapsulated SCE with a size of 80.46 ± 1.31 and an encapsulation efficiency of 79.18 ± 0.23 was formulated. At a concentration of 100 μg/ml, the greatest antimicrobial effect of the niosome-encapsulated SCE was correlated to Staphylococcus aureus, with an inhibition zone of 13.16 mm. The findings of the study revealed that all strains were unable to produce biofilms at a concentration of 100 μg/ml niosome-encapsulated SCE (p < 0.001). The survival rate of cancer cells after 72 h of exposure to niosome-encapsulated SCE was 40 ± 3.0%. Encapsulated SCE in niosomes inhibited cell progression in MCF-7 cells by increasing G0/G1 and decreasing S phase relative to G2/M phase; as a result, it activated the apoptosis signaling pathway and led to the induction of apoptosis in 69.12 ± 1.2% of tumor cells by increasing the expression of proapoptotic genes (p < 0.001). The results indicate that sea cucumber species from the Persian Gulf are a promising source of natural chemicals with antibacterial and anticancer properties, paving the path for novel marine natural products to be discovered. This is the first demonstration that niosome-encapsulated SCE contains sample was regenerated in 10 ml of 80% ethanol, homogenized for 2 min on ice with a professional Tissue-Terror, and subsequently centrifuged at 2500 rpm for 10 min at 4 • C. The supernatant was lyophilized after being filtered via a plastic film of 100 μm. The ethanol-extracted lyophilized substance was resuspended in PBS with 10% dimethyl sulfoxide [DMSO], vortexed, and centrifuged at 13,000 rpm for 10 min. The samples were filtered using 0.2 μm filters, and the resultant extracts, known as sea cucumber extract [SCE], were utilized in the experiment as stated.

Encapsulation of SCE in niosome
Niosome encapsulation of SCE was conducted through thin-film hydration. , China] in a 3:3:4 M ratios and suspended in 10 ml chloroform and methanol mixed in 2:1 ratio. After adding glass beads to the complex, the solvents were dried for 60 min at 60 • C and 120 rpm rotation using a rotary vacuum evaporator [Heidolph, Germany]. To generate a good niosome formulation, dried thin films were hydrated for 60 min at 60 • C at a speed of 120 rpm using a solution of SCE mixed in 10 ml of PBS. The obtained nanoparticle was centrifuged at 4 • C for 30 minto decrease the size of niosomes-SCE, and samples were kept at 4 • C for subsequent studies [49,50].

Niosome-encapsulated SCE morphological characteristics and entrapment efficiency [EE]
Using dynamic light scattering [DLS] and zeta-plas palladium electrodes, the uniform distribution, size distribution, and zeta potential of SCE loaded in niosomes were determined [Brookhaven Instruments Corp., USA]. The typical z diameter and multiple scattering index of niosomes were calculated, as well as their zeta potentials. To create electrical conductivity, niosome-encapsulated SCE particles were coated with a gold layer and analyzed with a MIRA3 field scanning electron microscope [FESEM] [TESCAN, Czech Republic]. A TEM apparatus with a magnification of 60,000× and a voltage of 26 kV was used to examine the morphology of the niosomes. The niosome was produced using a grid with a form covering after being treated with glutaraldehyde and then negatively stained with uranyl acetate. The quantity of noncapsulate SCE [free SCE] in the produced niosomes was measured to determine entrapment efficiency. Niosome-encapsulated SCEs were isolated in a refrigerated centrifuge at 14,000 rpm for 60 min. The EE % was calculated by Eq. (1), and the SCE concentration of the supernatant was determined using an ELISA Reader Stat Fax2100 [Awareness Technology, Ukraine] light absorbance measurement of the supernatant at 276 nm wavelength.
Dialysis was employed to examine SCE's release of the noisome. The dialysis tube was soaked in distilled water for 24 h. Then, 0.5 ml (10 mg) of SCE-loaded noisome was placed in a dialysis bag, and 0.5 ml SCE aqueous solution containing 10 mg SCE was also used as a control sample. Dialysis bags were immersed in conical flasks in 75 ml distilled water and shaken at 50 rpm in a water bath at 37 • C. Five milliliters were withdrawn from the receptor medium at intervals of 1, 2, 4, 6, 12, and 24 h, Aliquots of samples were replaced with a new medium at 37 • C, and imipenem was measured by spectrophotometry at 281 nm. The diffusion profile was determined using various kinetic models. This method was used to monitor the stability of diffusion of various Niosome-encapsulated SCE formulations at intervals of 7,14,21,28,35,42,49, and 56 days for a storage period of two months at 25 • C. as a solvent, 100 μg/ml niosome-encapsulated SCE was produced. Each plate had 50 μl of niosome-encapsulated SCE introduced into each well and incubated at 37 • C overnight. To determine the antibacterial and antifungal activities of niosome-encapsulated SCE, the average diameter of the inhibitory zones in millimeters was measured. For each substance under investigation, the average of the three replicates was computed. The positive control was ciprofloxacin dissolved in DMSO at 100 μg/cm 3 , while the negative control was sterile DMSO.

Biofilm growth inhibition [% BGI]
The efficacy of niosome-encapsulated SCE biofilm growth suppression by attachment to model biofilms was assessed using the Vyas [2007] technique [27], which involved evaluating the OD630 decrease of biofilms exposed to free SCE and niosome-encapsulated SCE for 2 h. Biofilm-containing wells were treated with 100 μg/ml niosome-encapsulated SCE in a 200 μl PBS solution. Control groups included free SCE and SCE combined with a blank niosome. The liquid components of each well were aspirated, and biofilms were cleaned with ethanol after 2 h of incubation at 37 • C. After that, antibiotic-free culture was introduced to the wells, and the biofilm and aspirate liquid contents were fixed and washed. Biofilms were subsequently dyed with crystal violet, their light absorbance was , 2 mM glutamine, and antibiotics [penicillin G, 60 mg/L; streptomycin, 100 mg/L; and amphotericin B, 50 mg/L] before being exposed to a humid and warm medium [37 • C, 5% CO2, 95%]. The standard solutions [100 mg/ml SCE in DMSO] were added to the medium containing enough volumes to obtain the desired concentrations and then cultivated with cells for 24 h, while the DMSO solution served as a blank reagent, for both niosome-encapsulated SCE and free SCE treatment.

Determination of cell viability
MTT assay was used to assess the vitality of MCF-7 cancer and HUVEC normal cell lines after treatment with niosome-encapsulated

Cell cycle analysis
The niosome-encapsulated SCE group, free SCE group, and blank group of MCF-7 cells were fixed for 24 h in absolute ethanol. Before being stained for 15 min with BD Bioscience Pharmingen's PI/RNase staining buffer, the cells were washed twice in PBS. FACS flow cytometry was used to determine the DNA content of the cell population. FlowJo version 10 software (https://www. bdbiosciences.com/en-eu/products/software/flowjo-v10-software) was used to evaluate cell cycle data [Tree Star, Ashland, OR].

Expression of apoptosis-related genes by quantitative real-time PCR
A quantitative real-time PCR approach with SYBR green detection was used to examine the expression of the proapoptotic genes FAS, BAK, BAX, and P53, as well as the anti-apoptotic genes BCL2 and SURVIVIN. The manufacturer's approach for obtaining RNA was TRIzol [Invitrogen, Carlsbad, CA]. Using the Transcript RT kit [Tiangen Biotech, Beijing, China], first-strand cDNA was created. Quantitative real-time PCR was carried out utilizing specific primers [ Table 1] and a SYBR® Premix Ex Taq™ II kit [TaKaRa, Japan] based on the manufacturer's instructions. A Rotor gene 6000 Corbett system was employed for amplification. Thermal cycling conditions were set as follows: an initial activation step for 5 min at 95 • C, followed by 40 cycles of 95 • C for 15 s and 60 • C for 1 min. Standard curves were created using data from serially diluted samples to confirm the reaction efficiencies of each primer set. Each primer set was also subjected to melting curve analysis. To verify the product size, PCR products were electrophoresed on a 1% agarose gel. GAPDH was employed to be the control.

Statistical analysis
All of the trials were carried out three times. The mean difference between groups was estimated using independent T test or analysis of variance [ANOVA] statistical techniques in the Statistical Package for Social Sciences [SPSS, Inc., Chicago, IL, USA] version 20 (https://www.ibm.com/support/pages/downloading-ibm-spss-statistics-20). GraphPad Prism version (https://www.bioz.com/ result/graphpad%20prism%207%200%20software/product/Graph%20Pad%20Software%20Inc) for Windows [GraphPad Software, USA] was used to create the graphs. All P values were less than 0.05, indicating that they were statistically significant.

Niosome-encapsulated SCE with a uniform spherical structure and low PDI index has high entrapment efficiency
Formulations of niosome-encapsulated SCE (100 μg/ml) were studied morphologically. The chemical composition of extracts of sea cucumber body organs was measured by GC-MS and recorded in Table S1. This formulation had a distinct size, polydispersity index [PDI], and entrapment efficiency [EE]. Dynamic light scattering [DLS] revealed that formulations of niosome-encapsulated SCE had good uniformity [ Table 2]. As demonstrated, this formulation of niosome-encapsulated SCE is of a smaller and better size and is associated with surfactant Span60's hydrophile-lipophile balance. Span60 has a hydrophile-lipophile balance of 4.7. Therefore, this nanoparticle formulated with Span60 is 80.46 ± 1.31 nm in size. In addition, the EE content of this formulation was 79.18 ± 0.23. The permeability of SCE in niosomes is directly related to the length of the saturated alkyl chain; thus, longer saturated alkyl chains result in higher permeability. Because Span60 has a long alkyl chain, formulations including it have higher indices, and the EE content is at its highest in this formulation. A lower polydispersity index [PDI] shows a suitable distribution of small nanoparticles, implying that such a formulation is the best since it has the lowest PDI. Fig. 1 shows that the niosome-encapsulated SCE of this formulation has a homogeneous spherical shape with an average size of 80.46 ± 1.31, indicating that the drug formulation's diameter [100 μg/ml] is acceptable. The particles have a spherical structure, as illustrated in the TEM diagram.

Niosome-encapsulated SCE Increases the antimicrobial and antibiofilm properties of SCE
The outcomes of 100 g/ml methanol extracts of niosome-mixtured SCE, niosome-encapsulated SCE, and free SCE were investigated. S. aureus was utilized as a bacterial species for the antibacterial and antifungal assays. The fungus species C. albicans was utilized. The niosome-encapsulated SCE had the best impact. It had the greatest inhibitory impact on the strains indicated. At a dosage of 100 μg/ml, the greatest effect of the niosome-encapsulated SCE was linked to the S. aureus strain, with an inhibition zone of 23.16 mm, while the inhibitory zone for C. albicans was 10.99 mm. The maximum inhibitory effect of the niosome-mixture SCE and free SCE was also on strains of S. aureus at a concentration of 100 μg/ml, with inhibition zones of 14.26 and 14.12 mm, respectively, while the maximum inhibitory effect was significant at a concentration of 100 μg/ml of the strain of C. albicans, with inhibition zones of 8.40 and 8.09 mm [ Table 3].
The microtiter plate technique was utilized to quantitatively examine the antibiofilm effects of all three groups. The findings in this study revealed that all strains were unable to produce biofilms at a concentration of 100 μg/ml niosome-encapsulated SCE, as indicated in Table 4. Other groups, on the other hand, have a comparable capacity to produce biofilms. However, biofilms formed in niosomemixed SCE groups, and free SCE was weak. Fig. 2 shows that following treatment with all three formulations [niosome-mixed SCE, niosome-encapsulated SCE, and free SCE], the light absorption of all strains decreased significantly [p < 0.001]. These findings revealed that bacteria and fungi proliferated at a slower rate, resulting in a lower quantity of cell mass and little or minimal biofilm development. Based on these findings, sea cucumber extract exhibits strong antibiofilm effects on both fungal and bacterial strains at a concentration of 100 μg/ml.

Niosome-encapsulated SCE reduces tumor cell proliferation by inducing apoptosis
Sea cucumber extract efficiency in MC7-7 cell lines was assessed. We gathered MCF-7 human breast cancer cell lines into 3 groups [niosome-mixed SCE, niosome-encapsulated SCE, and free SCE]. Cell viability was evaluated using MTT assays 24 h, 48 h, and 72 h after MCF-7 cells were grown in 96-well plates. Similarly, MTT experiments revealed that in the niosome-encapsulated SCE group,  Fig. 4]. In the niosome-encapsulated SCE group of MCF-7 cell lines, the proportion of apoptotic cells was substantially higher than in the niosome mixed SCE with and free SCE groups. Niosome-encapsulated SCE increased total apoptosis [69.12%] in MCF-7 cells, according to these findings. The proportion of total apoptosis in the two control groups, niosome mixed SCE and free SCE, was 61.12 and 55.58%, respectively.
Cell cycle advancement is linked to the acceleration of cell proliferation. Flow cytometry was used to examine cell cycle regulation in the niosome-encapsulated SCE group and control groups [niosome mixed SCE and free SCE] of MCF-7 cells [Fig. 5]. Sea cucumber extracts in the MCF-7 cells showed an increase in G0/G1 and a decrease in the S phase to G2/M phase ratio compared to the MCF-7 blank control cell. Sea cucumber extracts hindered cell cycle progression, according to these findings [ Fig. 5A]. For this study, proapoptotic P53, BAK, FAS, and BAX genes and antiapoptotic genes BCL2 and SURVIVIN were selected, and real-time PCR was performed to evaluate the expression of these genes in three MCF-7 cell lines of the niosome-encapsulated SCE group and control groups [niosome mixed SCE and free SCE]. As expected, proapoptotic gene expression was significantly higher in the niosome-encapsulated SCE groups   Fig. 5 shows that the expression of P53, BAK, BAX, and FAS apoptotic genes increased in the MCF-7 cell line treated with sea cucumber extracts. In contrast, the expression of BCL2 and SURVIVIN antiapoptotic genes showed a decrease in the MCF-7 cell line treated with sea cucumber extracts. As a result, treatment with sea cucumber extracts enhanced the production of apoptotic genes while decreasing the expression of antiapoptotic genes in the MCF-7 cancer cell line. In contrast, when MCF-7 blank cells were compared to cells in the treatment groups, apoptotic gene expression was found to be significantly higher, with a P < 0.001 significance level. These findings show that sea cucumber extract has a direct influence on the activation of apoptosis in this cell line. According to the results of this study, encapsulation of sea cucumber extract with niosome nanoparticles enhances the induction of apoptotic cells, which can be assessed at a significance level of P < 0.05.

Discussion
Sea cucumber is the most common in the Persian Gulf and is found across southern Iran [28]. Because of their great medicinal and nutritional potential, sea cucumbers are the most marketed and harvested of all echinoderms [29]. Sea cucumbers have been used in traditional medicine in Southeast Asia and the Middle East for many years to treat hypertension, bronchitis, arthritis, wounds, and constipation, among other ailments [30]. Although much research has been done on sea cucumber extracts to find powerful bioactive chemicals with putative anti-inflammatory, immunostimulatory, and anticancer effects, there have been relatively few investigations on sea cucumber bioactivities [31]. Given the huge number of bioactive chemicals found in sea cucumbers [32]. Many investigations on the antibacterial effects of a variety of marine animals, including Echinodermata, have been conducted in recent years by various countries [33].
Sea cucumber exhibited the highest antibacterial activity compared to other marine species, including Porifera, Bryozoa, Molluska, Coral, and Annelida [ringed worms] [34]. Methanol, aqueous methanol, ethanol, and chloroform extracts, as well as triterpene compounds, have been employed in the bulk of research on sea cucumber antibacterial and antifungal properties [8,35]. The antifungal activity of H. polii [a particular type of sea cucumber] in the Mediterranean Sea was studied using standard disc diffusion by certain researchers [35,36]. Research demonstrated that 2.5 mg/ml ethanolic extracts of the sea cucumber body wall exhibited significant antifungal activity against Aspergillus flavus, Aspergillus niger, and C. albicans [38]. According to the disk diffusion technique of many studies, the antimicrobial activities of the aqueous methanol extract of H. polii have a significant inhibitory impact on A. funmigatus and a lesser ability to inhibit Trichophyton rubram but no effect on C. albicans [39].
Sea cucumber extract has also been studied for its cancer-fighting properties [40]. The antitumorigenic effects of triterpene glycosides derived from sea cucumbers were investigated in an animal model of S180 sarcoma and mouse Lewis lung cancer cell lines [41]. In human colorectal cancer, an aqueous extract of sea cucumber was found to dramatically suppress proliferation and cause intense cytotoxicity in Caco-2 cells, a kind of intestinal cell [42,56]. In DLD-1, WiDr, and Caco-2 human colon carcinoma cells, extracted sphingoid bases of sea cucumber [Stichopus variegatus] demonstrated strong cytotoxic effects and reduced cell proliferation, as well as induction of apoptosis via caspase 3 activation [43]. Holothurin A [HA] and 24-dehydroechinoside A [DHEA], both sulfated triterpene glycosides derived from the sea cucumber genus [Pearsonothuria graeffei], were found to have antitumor and antimetastatic properties in research [44][45][46][47][48][49][50].
Niosomes have been the subject of an increasing number of studies in recent years due to the advancement of nanotechnologies in the pharmaceutics industry. Due to their capacity to encapsulate various medications to boost their stability and effectiveness, niosomes can be used as an alternative to liposomes and polymersomes. In contrast to other nanoparticles, liposomes, polymersomes, and   niosomes share a lot of structural commonalities and can all be filled with both hydrophilic and hydrophobic medications. They might therefore co-deliver hydrophilic and hydrophobic medications in the same vesicle. Niosomes have received a lot of attention due to their outstanding biocompatibility and comparatively low toxicity. Niosomes have advantages over liposomes, such as superior stability, low cost, simplicity in formulation, and scaling-up. Because non-ionic surfactants, which are the building blocks of niosomes, are more physically and chemically stable than lipids, niosomes are significantly more stable [53,54]. However, no research on the use of nanomaterials to improve sea cucumber extract has been conducted thus far [45][46][47][48][49][50][51][52][53][54][55][56]. This is one of the initial studies to investigate encapsulating sea cucumber extract by niosome nanoparticles. The encapsulating technique was used in this investigation after the sea cucumber extract was obtained. Fig. 1 shows a scanning electron microscopic picture of niosomes. Most niosomes have a circular form, with a size distribution of approximately 75.32 ± 0.34 nm or less, according to SEM and TEM examinations, which matches the results of dynamic light scattering experiments. However, the effective encapsulation of the extracts was shown by increasing the size of niosome nanostructures in the SCE encapsulating group [80.46 ± 1.31] while maintaining a homogeneous spherical structure. S. aureus and Candida albicans were inhibited by niosome-encapsulated SCE at doses of 100 μg/ml, according to our observations. Our findings show that antibacterial activity can be found in materials containing various components in the sea cucumber body. Furthermore, by encapsulating sea cucumber extract, niosome nanoparticles inhibit the molecular reactivity of the extract components with the growth medium, lowering the quality of the extract. Furthermore, niosomal nanoparticles improve drug delivery. The drug's adverse effects, such as toxicity and hemolysis, are lessened by niosome. Reduced side effects and a sharp rise in breast cancer cells were observed. The P-glycoprotein (P-gp) is efficiently inhibited by niosomes, increasing the bioavailability of anticancer and antiviral medications. To fight various illnesses, several pharmacological substances may be able to use niosomal drug delivery. By overcoming the anatomical barrier of the gastrointestinal system by transcytosis of M cells from Peyer's patches in the intestinal lymphatic tissues, it improves bioavailability. The reticuloendothelial system absorbs the niosomal vesicles. Such localized medication accumulation is employed to treat disorders where some issues infiltrate the liver and spleen cells [53,55].
As a result, sea cucumber extract encapsulated with niosomes dramatically boosted its antibacterial activity. As a result, sea cucumbers encapsulated in niosomes can be used as a source of antibacterial chemicals, making them suitable candidates for the production of pharmaceutical and medicinal compounds, as well as antibiotics. The effect of sea cucumber water extract [Sichopus variegatus] on rat spinal astrocyte cell lines was investigated by a researcher. Extracts (0.1, 1.0, 5.0, and 10.0 μg/ml) were produced.
Their findings imply a dose-dependent effect of S. aureus water extract on spinal astrocyte proliferation and differentiation. However, using a novel formulation of sea cucumber extract, our recent investigation found that it had superior antibacterial and antifungal properties. On day three after treatment with niosome-encapsulated SCE, MCF-7 cell proliferation was reduced, although normal cell viability was unaffected. By enhancing polarity, the addition of Span 60 to niosomes improved tumor cell specificity. As a result, encapsulating SCE in a niosome increased its anticancer activity in a particular way. Encapsulated sea cucumber extracts inhibited cell progression in MCF-7 cells by increasing G0/G1 and decreasing S phase relative to G2/M phase; as a result, it activated the apoptosis signaling pathway and led to the induction of apoptosis in 69.12% of tumor cells by increasing the expression of proapoptotic genes.
The results indicate that sea cucumber species from the Persian Gulf are a promising source of natural chemicals with anticancer properties, paving the path for novel marine natural products to be discovered. This is the first demonstration that niosome- encapsulated SCE contains antibacterial and anticancer chemicals that, according to their specific characteristics, boost antitumor activity. Further research is needed to purify and characterize the biological activity stated above.

Ethical approval
The authors of this article confirm that all methods were carried out in accordance with relevant guidelines and regulations. The authors of this article state that all methods are reported in accordance with ARRIVE guidelines (https://arriveguidelines.org). All animal protocols were performed in accordance with the Ethical Committee and Research Deputy of the Islamic Azad University of East-Tehran Branch, Iran for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee guidelines of Islamic Azad University, East-Tehran, Iran.

Author contribution statement
Tohid Piri-Gharaghie, Ghazal Ghajari, Neda Jegargoshe-Shirin: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data.
Maryam Hassanpoor: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.

Funding statement
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability statement
Data included in article/supp. Material/referenced in article.

Declaration of interest's statement
The authors declare no conflict of interest.